Glucocorticoid Receptor Nuclear Translocation Research Articles

Astragaloside IV inhibits microglia activation via glucocorticoid receptor mediated signaling pathway.

Inhibition of microglia activation may provide therapeutic treatment for many neurodegenerative diseases. Astragaloside IV (ASI) with anti-inflammatory properties has been tested as a therapeutic drug in clinical trials of China. However, the mechanism of ASI inhibiting neuroinflammation is unknown. In this study, we showed that ASI inhibited microglia activation both in vivo and in vitro. It could enhance glucocorticoid receptor (GR)-luciferase activity and facilitate GR nuclear translocation in microglial cells. Molecular docking and TR-FRET GR competitive binding experiments demonstrated that ASI could bind to GR in spite of relative low affinity. Meanwhile, ASI modulated GR-mediated signaling pathway, including dephosphorylation of PI3K, Akt, I κB and NF κB, therefore, decreased downstream production of proinflammatory mediators. Suppression of microglial BV-2 activation by ASI was abrogated by GR inhibitor, RU486 or GR siRNA. Similarly, RU486 counteracted the alleviative effect of ASI on microgliosis and neuronal injury in vivo. Our findings demonstrated that ASI inhibited microglia activation at least partially by activating the glucocorticoid pathway, suggesting its possible therapeutic potential for neuroinflammation in neurological diseases.

Blockade of interleukin-6 receptor enhances the anti-arthritic effect of glucocorticoids without decreasing bone mineral density in mice with collagen-induced arthritis.

In a mouse arthritis model, we investigated whether interleukin-6 receptor (IL-6R) blockade would enhance the anti-arthritic effect of glucocorticoids (GCs). DBA/1J mice were immunized with type II collagen (CII), and were treated with prednisolone (PSL) and/or anti-mouse IL-6R antibody (MR16-1). Also, the effects of IL-6 on gene expression and the nuclear translocation of glucocorticoid receptors (GRs) were examined in cultured cells treated with dexamethasone (DEX). PSL reduced the arthritis score dose-dependently in the collagen-induced arthritis (CIA) mouse model. The arthritis score in the PSL (3 mg/kg) + MR16-1 group was lower than in the PSL (3 mg/kg) group, and at the same level as in the PSL (6 mg/kg) group. Lumbar vertebra bone mineral density (BMD) was decreased significantly in CIA mice and was higher in the PSL (3 mg/kg) + MR16-1 group than in the PSL (6 mg/kg) group. In the in-vitro synovial cells, IL-6 pretreatment attenuated the inhibitory effect of DEX on cyclooxygenase (COX)-2 expression and inhibited the nuclear translocation of GR induced by DEX. In contrast, in MC3T3-E1 osteoblastic cells, IL-6 pretreatment exacerbated the decrease in expression of osteocalcin and the increase in expression of receptor activator of nuclear factor kappa-B ligand (RANKL) by DEX. We demonstrated that IL-6 signalling blockade by an anti-IL-6R antibody can augment the anti-arthritic effect of GCs and inhibit the bone loss they cause.

Abstract 4995: HO1 induction interrupts the glucocorticoid receptor signaling in prostate cancer cells

Abstract Although initially treatable, prostate cancer can recur in a hormone refractory form that is not responsive to current available therapies. The mortality rate associated with hormone refractory prostate cancer is high, and there is an urgent need for new therapeutic agents to treat prostate cancer. In order to overcome the toxicity of chemotherapy, glucocorticoids have long been used in the treatment of hormone-resistant prostate cancer. Nevertheless, many authors reported that the glucocorticoid receptor (GR) can substitute the androgen receptor and activate a similar but distinguishable set of genes. A common feature of prostate cancer is the dependence on activated signal transducer and activator of transcription 3 (STAT3), a transcription factor for survival and a coactivator of GR signaling. We have previously demonstrated that the induction of HO1, an antioxidant and anti-inflammatory protein, increases STAT3 cytoplasmic retention, interfering with its signaling and promotes HO1 nuclear localization. Our aim is to study the effect of HO1 induction on GR signaling in prostate cancer. PC3 cells were treated with Dexamethasone (Dex) at different concentrations (10−7, 10−8 and 10−9 M) during 24 h in 10% charcoaled-serum media and were exposed to the pharmacological inducer of HO1 (hemin, 80 uM, 24 h). Control cells were treated with vehicles. No significant differences in cell viability were detected by MTT assay. In order to investigate whether HO1 interrupts the GR transcriptional activity, PC3 cells were transfected with a luciferase reporter plamid containing three glucocorticoid response elements. Transfected cells were treated either with hemin (80 uM, 24 h), Dex (10−8 M, 6 h) or the combination of both drugs. The GR transcriptional activation was confirmed when cells were treated with the agonist Dex (p<0.01). HO1 induction significantly reduced either the GR basal transcriptional activity (p<0.01) as well as the Dex induced (p<0.01). The expression of Ikb, a GR regulated gene, was screened by RT-qPCR and was confirmed its inhibition in hemin exposed cells treated or not with dexamethasone. The repressive activity of HO1 was also demonstrated using an NFkB luciferase reporter assay. Using confocal fluorescence microscopy, GR nuclear translocation upon Dex treatment was confirmed, and a decrease of GR nuclear localization was demonstrated under hemin+Dex treatment (p<0.01). These results correlate with the diminished luciferase activity of the MMTV-luc plasmid in the presence of hemin+Dex vs Dex alone. Strikingly this is the first report showing the immunoprecipitation of GR and HO1, showing physical interaction between these proteins, both in the nucleus and in the cytoplasm. In conclusion, these results provide a novel function for HO1 downmodulating GR transcriptional activity, repressing the expression of its target genes and interfering with the receptor's nuclear localization in prostate cancer cells. Citation Format: Daiana B. Leonardi, Alejandra V. Paez, Federico Schuster, Nicolas Anselmino, Javier N. Brandani, Mercedes Abbate, Geraldine Gueron, Javier H. Cotignola, Elba S. Vazquez. HO1 induction interrupts the glucocorticoid receptor signaling in prostate cancer cells. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 4995. doi:10.1158/1538-7445.AM2015-4995

Ameliorative Effects of Neurolytic Celiac Plexus Block on Stress and Inflammation in Rats with Partial Hepatectomy

Purpose: To investigate effects of neurolytic celiac plexus block (NCPB) on stress and inflammation in rats with partial hepatectomy (PH).Methods: A model of PH rat was established, and serum C-reactive protein (CRP); corticosterone (GC); adrenocorticotropin (ACTH); noradrenaline (NA); adrenalin (AD); aspartate transaminase (AST); alanine transaminase (ALT); as well as tumor necrosis factor-α (TNF-α); interleukin (IL)-1β and IL-6; high mobility group box1 (HMGB1); and nitric oxide (NO) concentrations in serum assessed after PH. Additionally, Western blotting was performed to determine the effect of NCPB on expressions of glucocorticoid receptors (GR), inhibitor of nuclear factor kappa B (IκB), p65, c-Jun and inducible nitric oxide synthase (iNOS) of PH rats, as well as assay effects of NCPB on nuclear translocation of GR, c- Jun and p65. DNA binding activities of nuclear factor kappa B (NF-κB) and activator protein 1 (AP-1) were also determined.Results: NCPB reduced AST and ALT (P < 0.05), decreased secretion of inflammatory cytokines and NO (P < 0.05), as well as decreased CRP, GC, ACTH, NA and AD after PH (p < 0.05). NCPB increased expressions of GR and IκB, but expressions of p65, c-Jun, and iNOS (p < 0.05). Additionally, NCPB increased nuclear translocation of GR (p < 0.01), but decreased nuclear translocation of p65 and c-Jun after PH (p < 0.05). Additionally, DNA binding activity of NF-κB and AP-1 was decreased by NCPB (p < 0.05).Conclusion: The results indicate that NCPB treatment can significantly inhibit stress and inflammation in PH rats.Keywords: Neurolytic celiac plexus block, Cytokine, Nuclear translocation, Partial hepatectomy, Stress, Inflammation

Enhancing nuclear translocation: perspectives in inhaled corticosteroid therapy.

Corticosteroids are widely used in the treatment of asthma and chronic obstructive pulmonary disease (COPD). In contrast to their use in mild-to-moderate asthma, they are less efficacious in improving lung function and controlling the underlying inflammation in COPD. In most clinical trials, corticosteroids have shown little benefit in COPD, but have shown a greater clinical effect in combination with long-acting bronchodilators. Impaired corticosteroid activation of the glucocorticoid receptor (GR) has been reported in corticosteroid-insensitive individuals. Reversal of corticosteroid-insensitivity by enhancing GR nuclear translocation is a potential therapeutic target. Preclinical studies suggest members of the nuclear receptor superfamily may facilitate glucocorticoid receptor nuclear translocation. Unravelling the mechanisms that govern GR nuclear translocation may identify novel therapeutic targets for reversing corticosteroid-insensitivity.

MicroRNA-9 regulates steroid-resistant airway hyperresponsiveness by reducing protein phosphatase 2A activity

Steroid-resistant asthma is a major clinical problem that is linked to activation of innate immune cells. Levels of IFN-γ and LPS are often increased in these patients. Cooperative signaling between IFN-γ/LPS induces macrophage-dependent steroid-resistant airway hyperresponsiveness (AHR) in mouse models. MicroRNAs (miRs) are small noncoding RNAs that regulate the function of innate immune cells by controlling mRNA stability and translation. Their role in regulating glucocorticoid responsiveness and AHR remains unexplored. IFN-γ and LPS synergistically increase the expression of miR-9 in macrophages and lung tissue, suggesting a role in the mechanisms of steroid resistance. Here we demonstrate the role of miR-9 in IFN-γ/LPS-induced inhibition of dexamethasone (DEX) signaling in macrophages and in induction of steroid-resistant AHR. MiRNA-9 expression was assessed by means of quantitative RT-PCR. Putative miR-9 targets were determined in silico and confirmed in luciferase reporter assays. miR-9 function was inhibited with sequence-specific antagomirs. The efficacy of DEX was assessed by quantifying glucocorticoid receptor (GR) cellular localization, protein phosphatase 2A (PP2A) activity, and AHR. Exposure of pulmonary macrophages to IFN-γ/LPS synergistically induced miR-9 expression; reduced levels of its target transcript, protein phosphatase 2 regulatory subunit B (B56) δ isoform; attenuated PP2A activity; and inhibited DEX-induced GR nuclear translocation. Inhibition of miR-9 increased both PP2A activity and GR nuclear translocation in macrophages and restored steroid sensitivity in multiple models of steroid-resistant AHR. Pharmacologic activation of PP2A restored DEX efficacy and inhibited AHR. MiR-9 expression was increased in sputum of patients with neutrophilic but not those with eosinophilic asthma. MiR-9 regulates GR signaling and steroid-resistant AHR. Targeting miR-9 function might be a novel approach for the treatment of steroid-resistant asthma.

Impaired nuclear translocation of the glucocorticoid receptor in corticosteroid-insensitive airway smooth muscle in severe asthma.

Patients with severe asthma (SA) are less responsive to the beneficial effects of corticosteroid (CS) therapy, and relative CS insensitivity has been shown in airway smooth muscle cells (ASMC) from patients with SA. We investigated whether there was a defect in the actions of the glucocorticoid receptor (GR) underlying the ability of CS to suppress the inflammatory response in ASMC of patients with SA. ASMC from healthy subjects (n = 10) and subjects with severe (n = 8) and nonsevere asthma (N-SA; n = 8) were cultured from endobronchial biopsies. GR expression in ASMC from SA and N-SA was reduced compared with that from healthy subjects by 49% (P < 0.01). Although baseline levels of nuclear GR were similar, GR nuclear translocation induced by dexamethasone (10(-7) M) in SA was 60% of that measured in either healthy subjects or subjects with N-SA. Tumor necrosis factor (TNF)-α induced greater nuclear factor (NF)-κB (p65) mRNA expression in ASMC from subjects with SA (5.6- vs. 2.0-fold; P < 0.01), whereas baseline and TNF-α-induced nuclear translocation and dexamethasone-mediated suppression of p65 expression were similar between groups. Dexamethasone, although not modulating TNF-α-induced p65 nuclear translocation, attenuated p65 recruitment to the CCL11 promoter in the healthy and N-SA groups, but this suppressive effect was impaired in subjects with SA. Decreased GR expression with impaired nuclear translocation in ASMC, associated with reduced dexamethasone-mediated attenuation of p65 recruitment to NF-κB-dependent gene promoters, may underlie CS insensitivity of severe asthma.

Discovery of a novel isoxazoline derivative of prednisolone endowed with a robust anti-inflammatory profile and suitable for topical pulmonary administration

A novel glucocorticoids series of (GCs), 6α,9α-di-Fluoro 3-substituted C-16,17-isoxazolines was designed, synthesised and their structure–activity relationship was evaluated with glucocorticoid receptor (GR) binding studies together with GR nuclear translocation cell-based assays. This strategy, coupled with in silico modelling analysis, allowed for the identification of Cpd #15, an isoxazoline showing a sub-nanomolar inhibitory potency (IC50=0.84nM) against TNFα-evoked IL-8 release in primary human airways smooth muscle cells. In Raw264.7 mouse macrophages, Cpd #15 inhibited LPS-induced NO release with a potency (IC50=6nM)>10-fold higher with respect to Dexamethasone. Upon intratracheal (i.t.) administration, Cpd #15, at 0.1μmol/kg significantly inhibited and at 1μmol/kg fully counteracted eosinophilic infiltration in a model of allergen-induced pulmonary inflammation in rats. Moreover, Cpd #15 proved to be suitable for pulmonary topical administration given its sustained lung retention (t1/2=6.5h) and high pulmonary levels (>100-fold higher than plasma levels) upon intratracheal administration in rats. In summary, Cpd #15 displays a pharmacokinetic and pharmacodynamic profile suitable for topical treatment of conditions associated with pulmonary inflammation such as asthma and COPD.

P26 P38 MAPK Inhibition Enhances Corticosteroid Effects In Human Epithelial Cells Via Increased Gr Nuclear Localisation

Background Phospho-p38 MAPK expression is increased in airway epithelial cells in patients with severe asthma and in COPD patients compared to controls. Increased p38 MAPK activation may lead to reduced corticosteroid responsiveness. We have used a human cell bronchial epithelial cell line to investigate the effects of a p38 MAPK inhibitor in combination with a corticosteroid on inflammatory cytokine stimulation. Methods The human epithelial cell line 16HBE14o - was used to determine the effects of dexamethasone (0.1–1000 nM) alone, the p38 MAPK inhibitor BIRB-796 (1–1000 nM) alone and both drugs combined at all concentrations on LPS (1 µg/ml), Poly I:C (100 µg/ml) or TNFα (10 ng/ml) -induced IL-6, IL-8 and RANTES. 16HBE14o - cells were treated with BIRB-796 (1–1000 nM) alone and in combination with dexamethasone (0.1 nM) for 30 min and glucocorticoid receptor (GR) nuclear translocation determined by immunofluorescence. The effects of TNFα stimulation on the phosphorylation of p38 and GR (serine 226) in 16HBE14o - cells were determined by Western blot analysis. Results Maximum inhibition of dexamethasone and BIRB-796 in combination was significantly greater than either drug alone for LPS and TNFα induced IL-6 and IL-8 and for Poly I:C induced RANTES (p Conclusion P38 MAPK inhibition enhances the effect of corticosteroids on inflammatory cytokines in human epithelial cells. This enhancement is due to inhibition of p38 dependent phosphorylation of GR serine 226 which leads to increased nuclear localisation of GR.

25-Hydroxyvitamin D3-deficiency enhances oxidative stress and corticosteroid resistance in severe asthma exacerbation.

Oxidative stress plays a significant role in exacerbation of asthma. The role of vitamin D in oxidative stress and asthma exacerbation remains unclear. We aimed to determine the relationship between vitamin D status and oxidative stress in asthma exacerbation. Severe asthma exacerbation patients with 25-hydroxyvitamin D3-deficiency (V-D deficiency) or 25-hydroxyvitamin D-sufficiency (V-D sufficiency) were enrolled. Severe asthma exacerbation with V-D-deficiency showed lower forced expiratory volume in one second (FEV1) compared to that with V-D-sufficiency. V-D-deficiency intensified ROS release and DNA damage and increased TNF-α, OGG1 and NFκB expression and NFκB phosphorylation in severe asthma exacerbation. Supplemental vitamin D3 significantly increased the rates of FEV1 change and decreased ROS and DNA damage in V-D-deficiency. Vitamin D3 inhibited LPS-induced ROS and DNA damage and were associated with a decline in TNF-α and NFκB in epithelial cells. H2O2 reduces nuclear translocation of glucocorticoid receptors in airway epithelial cell lines. V-D pretreatment enhanced the dexamethasone-induced nuclear translocation of glucocorticoid receptors in airway epithelial cell lines and monocytes from 25-hydroxyvitamin D3-deficiency asthma patients. These findings indicate that V-D deficiency aggravates oxidative stress and DNA damage, suggesting a possible mechanism for corticosteroid resistance in severe asthma exacerbation.

Mucin 1 downregulation associates with corticosteroid resistance in chronic rhinosinusitis with nasal polyps

A number of patients with chronic rhinosinusitis with nasal polyps (CRSwNP) are resistant to oral corticosteroids. Mucin 1 (MUC1) shows anti-inflammatory properties, and its cytoplasmic tail (CT) interacts with transcription factors, facilitating their nuclear translocation. Because glucocorticoid receptor (GR) nuclear translocation is key to the anti-inflammatory effect of corticosteroids, we hypothesized that MUC1 is involved in the effectiveness of corticosteroids. To analyze the role of MUC1 in corticosteroid effectiveness in different cohorts of patients with CRSwNP and elucidate the possible mechanisms involved. Seventy-three patients with CRSwNP took oral corticosteroids for 15 days. Corticosteroid resistance was evaluated by nasal endoscopy. The expression of MUC1 and MUC1 CT was evaluated by real-time PCR, Western blotting, and immunohistochemistry. Beas-2B knockdown with RNA interference for MUC1 (siRNA-MUC1) was used to analyze the role of MUC1 in the anti-inflammatory effects of dexamethasone. Nineteen patients had nasal polyps that were resistant to oral corticosteroids (NP-CR). MUC1 expression was downregulated in these patients. Primary epithelial cells from patients with NP-CR were insensitive to the anti-inflammatory effects of dexamethasone. In siRNA-MUC1 Beas-2B, dexamethasone showed weaker anti-inflammatory effects, a reduced inhibition of phospho-extracellular-signal-regulated kinases 1/2, a less severe mitogen-activated protein kinase phosphatase 1 increase, and a reduced GR nuclear translocation. Immunoprecipitation experiments revealed that MUC1-CT and GRα form protein complexes and translocate to the nucleus in response to dexamethasone. MUC1-CT-GRα complex was downregulated in NP-CR tissue. MUC1-CT participates in the corticosteroid response that mediates GRα nuclear translocation. The low expression of MUC1 in patients with CRSwNP may participate in corticosteroid resistance.

Identification of a selective glucocorticoid receptor ligand for the treatment of chronic inflammation in type 2 diabetes mellitus.

The present study aimed to identify a new selective glucocorticoid receptor (GR) ligand for the treatment of chronic inflammation in type 2 diabetes mellitus. The IN Cell Analyzer 1000 platform was employed to screen for compounds that may promote GR nuclear translocation. A mammalian two-hybrid system and transactivation assay-were used to analyze the selected GR ligands and evaluate their activities for GR transcription and the recruitment of co-activators. A novel selective GR ligand, compound Q40, was identified that was able to promote GR nuclear translocation in a short period of time. It increased the ability of GR to recruit co-activators in a concentration-dependent manner, but had no positive effect on GR transcriptional activity. In conclusion, an increase in the expression levels of gluconeogeneic genes, induced by the transcriptional activation of GR, is the predisposing factor most commonly associated with the side-effects of glucocorticoids. The results suggest that compound Q40 is a ligand of the GR and exerts an agonistic action on the recruitment of co-activators without sugar dysmetabolism-related side-effects. Thus, compound Q40 has the potential to be used as an anti-inflammatory adjuvant therapy with minimal side-effects in patients with type 2 diabetes mellitus.

Expression and nuclear translocation of glucocorticoid receptors in type 2 taste receptor cells

Stress increases the secretion of glucocorticoids (GCs), potent steroid hormones that exert their effects on numerous target tissues by acting through glucocorticoid receptors (GRs). GC signaling significantly affects ingestive behavior and taste preferences in humans and rodent models, but far less is known about the hormonal modulation of the peripheral sensory system that detects and assesses nutrient content of foods. A previous study linked restraint stress in rats to diminished expression of mRNA for one subunit of the sweet taste receptor (Tas1r3) in taste tissue and reduced gustatory nerve excitation by sweet compounds. Using RT-PCR, we detected mRNAs for GRα in circumvallate taste papillae and in oral epithelium devoid of taste buds (“non-taste” tissue). Further, circumvallate tissue was significantly enriched in GR mRNA compared to non-taste tissue based on quantitative PCR. Histologically, GR protein was expressed in all taste bud populations examined (circumvallate, foliate and fungiform papillae). Using transgenic mice expressing green fluorescent protein, almost all (97%) Tas1r3-positive taste cells (sweet-/umami-sensitive) expressed GR compared to a significantly smaller percentage (89%) of TrpM5-positive taste cells (sweet-, umami- and bitter-sensitive). When mice (n=4) were restrain stressed, GR protein mobilized to the nucleus in Tas1r3-GFP taste cells (1.7-fold over controls). Our results suggest that GR can be activated in taste receptor cells and may play a role in specific taste qualities (e.g., sweet, umami, and bitter) to shape how the taste system responds to stress.

FGFR4 Polymorphic Variants Modulate Phenotypic Features of Cushing Disease

Cushing disease is a potentially lethal condition resulting from hormone excess, usually due to a small pituitary tumor that fails to respond to negative feedback inhibition. A minority of patients develop larger, more aggressive tumors of the same lineage but with modest hormone excess. Here we show that a common polymorphism in the fibroblast growth factor receptor 4 (FGFR4) transmembrane domain yields receptor isoforms with distinct properties that mediate these biological differences. Forced expression of the major FGFR4-G388 variant allele supports pY-signal transducer and activator of transcription (STAT3) responses. In contrast, expression of the minor FGFR4-R388 allele enhances STAT3 serine phosphorylation, driving cellular growth. In addition, FGFR4-R388 enhances glucocorticoid receptor phosphorylation and nuclear translocation. Consistent with these findings, glucocorticoid administration resulted in enhanced hormone negative feedback in mice with knock-in of the FGFR4 variant allele. Moreover, clinical data from patients with pituitary tumors revealed that those homozygous for the R388 allele have a higher frequency of silent corticotroph macroadenomas than FGFR4-G388 carriers, who were more likely to have small but hormonally active microadenomas. These findings demonstrate that the FGFR4 transmembrane polymorphic variants can modulate cellular growth and sensitivity to glucocorticoid hormone negative feedback through distinct STAT3 modifications of relevance to the human forms of Cushing disease.

Lose dose genistein inhibits glucocorticoid receptor and ischemic brain injury in female rats

Although acute bolus of genistein treatment has been shown to protect against neuronal damage in experimental brain injury animal models, chronic continuous low dose treatment of genistein on ischemic brain injury has not been well elucidated. In the present study, female rats were received either pure genistein (0.1mg/kg/day via osmotic minipumps) or placebo at the time of ovariectomy, and transient forebrain ischemia was induced 7days later. Results demonstrated that genistein treatment for 14days significantly improved ischemic neuronal survival in hippocampal CA1 region of ovariectomized rats. Glucocorticoid receptor (GR) is a member of the adrenal steroid hormone receptor, which is highly expressed in the rat hippocampus. Activation of the GR plays a critical role in the neuronal stress responses, including ischemic brain damage. This study therefore examined the potential mechanisms by which genistein regulates GR signaling, including the protein distribution and receptor activation in hippocampus following ischemic reperfusion (I/R). Results showed that GR expression in the ovariectomized rats was excessively increased both in neurons (I/R 6h) and activated microglial cells (I/R 7d) in hippocampal CA1 region. Genistein treatment significantly attenuated GR induction and the enhanced GR nuclear translocation and DNA-binding capacity. The effects of genistein on the GR levels was accompanied with decreased blood plasma levels of corticosterone (primary glucocorticoid in rodents) and coupled to an E3 ubiquitin ligase Mdm2 targeted proteasomal degradation of GR, because genistein treatment could enhance the GR–Mdm2 interaction and the ubiquitination level of GR protein. In addition, our results indicated that genistein markedly prevented the excessive activation of microglia in CA1 sector. These results demonstrate the neuroprotective action of chronic low dose genistein replacement against ischemic brain damage, and a potential mechanism associated with the inhibition of both neuronal and microglial GR signaling following ischemic stress.

S66 Targeting anti-ageing molecule AMPK restores corticosteroid sensitivity in COPD

RationaleChronic obstructive pulmonary disease (COPD) is an irreversible inflammatory lung disease and is currently the fourth greatest burden of disease of worldwide. However a key issue is that patients show...

Abstract C28: Dexamethasone-induced apoptosis in multiple myeloma cells is restricted to t(14;16) and t(4;14) molecular subgroups due to a combined down-regulation of MAF and an up-regulation of Bim.

Abstract Multiple myeloma (MM) is heterogeneous with respect to its causative chromosomal abnormalities and the treatment response of patients. Briefly, MM patients with chromosomal hyperdiploidy or those carrying a t(11.14) translocation have a better prognosis than those with a t(4;14) or t(14;16) translocation. Dexamethasone (Dex) is widely used in all phases of the treatment of MM as induction, consolidation or maintenance. However, the mechanisms of sensitivity and resistance to Dex still remain elusive. In the present study, we analyzed the ability of Dex to induce cell death by Apo-2.7 staining in 33 human myeloma cell lines (HMCLs) representative of the molecular subsets i.e., carrying a t(11;14), t(4;14) or t(14;16) translocation, which deregulates the expression of CCND1, MMSET, and c-MAF respectively. The mechanisms controlling Dex-induced apoptosis were evaluated by analyzing the nuclear translocation of glucocorticoid receptor (GR), signalling pathways and modulation of Bcl-2 protein family in the different MM molecular subtypes. We first demonstrated by transcriptomic analysis (Affymetrix) that GR expression was heterogeneous among the different molecular subtypes of HMCLs and patients. Indeed, t(14;16) c-MAF subgroup significantly expressed higher levels of GR than all other subgroups in both HMCLs (Kruskall-Wallis p&amp;lt;.02) and primary MM cells from patients at diagnosis (n=309, Kruskall-Wallis p&amp;lt;.0001). We further demonstrated that the direct pro-apoptotic effect of Dex was related to the genetic heterogeneity of MM, since sensitive HMCLs were restricted to t(14;16) and t(4;14) subgroups. Dex-induced apoptosis was associated with caspase 6 and 3 activation. We next demonstrated that Dex sensitivity depended on its ability to induce the down-regulation of c-MAF protein, which led to a decrease in CCND2 expression, a well-known target gene of c-MAF. The down-regulation of c-MAF mainly occurred by the increase of its proteasomal degradation. Altogether, these results were in agreement with the elevated levels of c-MAF found not only in t(14;16) translocated primary cells and HMCLs but also in t(4;14) translocated cells and HMCLs. While Bim expression (all isoforms) was up-regulated in most of cell lines by Dex irrespectively of its capacity to induce apoptosis, we demonstrated by Bim silencing that its up-regulation was required for initiating Dex-induced apoptotic program. Of note, by a long-term culture in the presence of low doses of Dex, we generated a Dex-resistant t(14;16) which was characterized by a decreased expression of GR. Dex treatment of this Dex-resistant HMCL neither induced any down-regulation of c-MAF nor any up-regulation of Bim, showing that a minimal expression level of GR was the first limiting step to induce a death response. In the present study, we demonstrated that Dex exerted a direct anti-tumor effect on myeloma cells from t(14;16) and t(4;14) molecular subgroups by controlling c-MAF level while those belonging to the t(11;14) subgroup were insensitive to Dex. This result highlights the fact that conventional therapeutic approaches could be re-evaluated in concert with the definition of molecular subgroups of MM patients to favor molecular subtype-specific therapy based on rational preclinical data. Citation Information: Mol Cancer Ther 2013;12(11 Suppl):C28. Citation Format: Charlotte Kervoëlen, Emmanuelle Ménoret, Patricia Gomez-Bougie, Régis Bataille, Catherine Pellat-Deceunynck, Martine Amiot. Dexamethasone-induced apoptosis in multiple myeloma cells is restricted to t(14;16) and t(4;14) molecular subgroups due to a combined down-regulation of MAF and an up-regulation of Bim. [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2013 Oct 19-23; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2013;12(11 Suppl):Abstract nr C28.

Inhaled long-acting β2 agonists enhance glucocorticoid receptor nuclear translocation and efficacy in sputum macrophages in COPD

Combination inhaled therapy with long-acting β2 agonists (LABAs) and corticosteroids is beneficial in treating asthma and chronic obstructive pulmonary disease (COPD). In asthma, LABAs enhance glucocorticoid receptor (GR) nuclear translocation in the presence of corticosteroids. Whether this biological mechanism occurs in COPD, a relatively corticosteroid-resistant disease, is uncertain. Eight patients with mild/moderate COPD participated in a double-blind, placebo-controlled, crossover study and inhaled single doses of fluticasone propionate (FP) 100 μg, FP 500 μg, salmeterol xinafoate (SLM) 50 μg, and combination FP 100 μg + SLM 50 μg. One hour postinhalation, sputum was induced, nuclear proteins isolated from purified macrophages, and levels of activated nuclear GR quantified by using a GR-glucocorticoid response element ELISA-based assay. Nuclear GR significantly increased after the inhalation of FP 500 μg (P < .01), but not after the inhalation of FP 100 μg or SLM 50 μg, compared with placebo. Interestingly, SLM in combination with FP 100 μg increased nuclear GR levels equivalent to those of FP 500 μg alone. This was significantly greater than either FP 100 μg (P < .05) or SLM 50 μg (P < .01) alone. In vitro in a human macrophage cell line, SLM (10(-8) mol/L) enhanced FP (10(-9) mol/L)-induced mitogen-activated protein kinase phosphatase-1 mRNA (5.8 ± 0.6 vs 8.4 ± 1.1 × 10(-6) copies, P < .05) and 2 × glucocorticoid response element-luciferase reporter gene activity (250.1 ± 15.6 vs 103.1 ± 23.6-fold induction, P < .001). Addition of SLM (10(-9) mol/L) to FP (10(-11) mol/L) significantly enhanced FP-mediated suppression of IL-1β-induced CXCL8 (P < .05). Addition of SLM 50 μg to FP 100 μg enhanced GR nuclear translocation equivalent to that seen with a 5-fold higher dose of FP in sputum macrophages from patients with COPD. This may account for the superior clinical effects of combination LABA/corticosteroid treatment compared with either as monotherapy observed in COPD.

Basal p38 Mitogen-Activated Protein Kinase Regulates Unliganded Glucocorticoid Receptor Function in Airway Smooth Muscle Cells

Like many steroid receptors, the glucocorticoid (GC) receptor (GR) is a phosphoprotein. Although there are multiple phosphorylation sites critical for GR transcriptional activity (i.e., serine [S]203, S211, and S226), their respective role in driving GR functions is highly cell specific. We have recently identified protein phosphatase 5 as an essential Ser/Thr phosphatase responsible for impairing GR function via S211 dephosphorylation in airway smooth muscle (ASM) cells. Because p38 mitogen-activated protein kinase (MAPK) directly phosphorylates GR in different cell types in a stimulus- and cell-dependent manner, we investigated the role of p38 MAPK on GR phosphorylation and function in ASM cells. Cells were transfected with 100 nM p38 MAPK small interfering RNA or 2 μg MAPK kinase 3 expression vector (a specific kinase that directly activates p38 MAPK) in the presence or absence of fluticasone (100 nM) and/or p38 MAPK pharmacological inhibitor SB203580. We found that p38 MAPK blockade positively regulates GR nuclear translocation and GR-dependent induction of the steroid-target gene GC-induced leucine zipper in a hormone-independent manner. We also found that p38 MAPK-dependent regulation of GR functions was associated with a differential action on GR phosphorylation at S203 and S211 residues. This study demonstrated that the inactive state of GR in resting conditions is not only ensured by the absence of the GC ligand but also by p38 MAPK-dependent phosphorylation of unliganded GR at specific residues, which appears to be important in determining the overall GC responsiveness of ASM cells.

Vaccination inhibits TLR2 transcription via suppression of GR nuclear translocation and binding to TLR2 promoter in porcine lung infected with Mycoplasma hyopneumoniae

Toll-like receptors (TLRs) and glucocorticoid receptor (GR) act respectively as effectors of innate immune and stress responses. The crosstalk between them is critical for the maintenance of homeostasis during the immune response. Vaccination is known to boost adaptive immunity, yet it remains elusive whether vaccination may affect GR/TLR interactions following infection. Duroc×Meishan crossbred piglets were allocated to three groups. The control group (CC) received neither vaccination nor infection; the non-vaccinated infection group (NI) was artificially infected intratracheally with Mycoplasma hyopneumoniae (M. hyopneumoniae); while the vaccinated, infected group (VI) was vaccinated intramuscularly with inactivated M. hyopneumoniae one month before infection. The clinical signs and macroscopic lung lesions were significantly reduced by vaccination. However, vaccination did not affect the concentration of M. hyopneumoniae DNA in the lung. Serum cortisol was significantly decreased in both NI and VI pigs (P<0.01), but only VI pigs demonstrated significantly diminished nuclear GR content. TLRs 1–10 were all expressed in lung, among which TLR2 was the most abundant and was significantly up-regulated (P<0.05) in NI pigs, but not in VI pigs. Accordingly, GR binding to the GR response element on TLR2 promoter was significantly increased (P<0.05) in NI pigs, but not in VI pigs. These results suggest that the inhibition of GR nuclear translocation and binding to the TLR2 promoter, which results in diminished TLR2 expression, is associated with the protective effect of vaccination on M. hyopneumoniae-induced lung lesions in the pig.